The mechanism of p53-mediated apoptosis remains poorly understood. Current evidence suggests that p53 induces cell death by separate transcription-dependent and -independent pathways. Mitochondrial involvement is critical for most forms of cell death. Data from the investigator's laboratory suggests that in cells with an intact p53- mediated apoptotic pathway, p53 protein is physically present within the mitochondria in a specific low-abundance protein complex with mt-hsp 70. Mt-hsp70 likely mediates mitochondrial import and refolding of p53. This apparent organellar location strongly argues for a direct transcription- independent regulatory role of p53 in the mitochondrial phase of apoptosis. In contrast, neuroblastoma cells which are resistant to apoptosis under the conditions tested, appear to contain abnormally high (deregulated) levels of mitochondrial p53/mt-hsp70 complexes, associated with covalent modification of p53. This suggests an altered function of p53 which may result in an anti-apoptotic phenotype at the mitochondrial level in this tumor. Biochemical and genetic experiments addressing the functional significance of p53's presence within mitochondria under physiological conditions are the core of this proposal.